During the past year we have demonstrated that Escherichia coli growth, both in aerobic and in anaerobic cultures, was under CO2 regulation. The rate of growth was proportional to CO2 concentration with saturation occurring at 0.02-0.03% CO2. The influence of CO2 on growth was also demonstrated with eucaryotic cells represented by normal (WI-38) and transformed (WI-18) embryonic lung cell lines. Each of the mammalian cell lines had dramatically different optimal CO2 and O2 concentrations. By culturing WI-38 under the appropriate gas atmosphere, we were able to produce cultures from inocula several orders of magnitude smaller than was formerly possible, and we successfully doubly cloned WI-38 in successive passages. The CO2 effect on growth of several bacteria reported previously and the examples above constitute a phylogenetic and nutritionally diverse sample of organisms. Each type of cell responded similarly. In every instance, CO2 (bicarbonate) was required to initiate growth. Once initiated, growth continued to be CO2 (bicarbonate) dependent. It is particularly significant that the rate of growth was regulated by the concentration of CO2 (bicarbonate). The uniform response suggests the phenomenon of CO2 regulation is a general one. The objective of the project is to identify the metabolic reaction controlled by CO2 (bicarbonate) and determine the mechanism by which this reaction is capable of regulating growth.